This research will be done primarily in Santiago, Chile at the University of Chile in collaboration with Drs. Segura-Aguilar and Caviedes as an extension of NIH grant # RO01 ES05865. The experimental goals described in this FIRCA application address fundamental mechanisms underlying the repair of DNA damage induced by reactive species in the dopaminergic systems and its possible relationship to Parkinson's disease. They also serve as the foundation for further expanding mutually beneficial collaborative research and training activities in DNA damage by reactive species shared by investigators at the University of South Alabama at College of Medicine and The Faculty of Medicine of University of Chile, Santiago,Chile. Specifically, the theme of the parent grant "Repair of DNA Damage induced by Environmental Agents" addresses the hypothesis that an acceptable lesion equilibrium is maintained within the mtDNA of cells through maintenance of a balance between mtDNA damage and mtDNA repair. If this lesion equilibrium is shifted such that the damage exceeds the repair capacity, fewer functioning mitochondrial genomes will be available for transcription and cellular bioenergetics will decrease. The cell will then either adapt to accommodate to this change in lesion equilibrium by decreasing cellular functions or, if the damage becomes excessive, mitochondrial function will cease and the cell die by either apoptotic or necrotic mechanisms. The opportunity to collaborate with Drs. Segura-Aguilar and Caviedes laboratories allows us to test our hypothesis in a model where reactive species are formed during reductive metabolism of dopamine oxidation products such as aminochrome. A plausible mechanism for aminochrome neurotoxicity is that it is mediated by mtDNA damage which induces apoptosis. To test this hypothesis, a neuronal cell line derived from rat substantia nigra (RCSN-3) will be utilized. The hypothesis will be pursued through the following specific aims: 1) To determine whether aminochrome one-electron metabolism induces mtDNA damage; 2) To determine whether aminochrome toxicity is mediated by DNA damage and apoptosis; 3) To determine whether DT-diaphorase, which has been proposed to be a neuroprotective enzyme prevents mtDNA damage in RCSN-3 cells during aminochrome metabolism. Thus, these specific aims are designed to enhance a central theme in our research which is to determine how different populations of neurons and glial cells and respond to damage to mtDNA caused by reactive oxygen species.